Bombesin inhibits alveolarization and promotes pulmonary fibrosis in newborn mice

RATIONALE: Bombesin-like peptides promote fetal lung development. Normally, levels of mammalian bombesin (gastrin-releasing peptide [GRP]) drop postnatally, but these levels are elevated in newborns that develop bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by arrested alveolarization. In premature baboons with BPD, antibombesin antibodies reduce lung injury and promote alveolarization. OBJECTIVES: The present study tests whether exogenous bombesin or GRP given perinatally alters alveolar development in newborn mice. METHODS: Mice were given peptides intraperitoneally twice daily on Postnatal Days 1-3. On Day 14 lungs were inflation-fixed for histopathologic analyses of alveolarization. MEASUREMENTS AND MAIN RESULTS: Bombesin had multiple effects on Day 14 lung, when alveolarization was about half complete. First, bombesin induced alveolar myofibroblast proliferation and increased alveolar wall thickness compared with saline-treated control animals. Second, bombesin diminished alveolarization in C57BL/6 (but not Swiss-Webster) mice. We used receptor-null mice to explore which receptors might mediate these effects. Compared with wild-type littermates, bombesin-treated GRP receptor (GRPR)-null mice had increased interstitial fibrosis but reduced defects in alveolarization. Neuromedin B (NMB) receptor-null and bombesin receptor subtype 3-null mice had the same responses as their wild-type littermates. GRP had the same effects as bombesin, whereas neither NMB nor a synthetic bombesin receptor type 3 ligand had any effect. All effects of GRP were abrogated in GRPR-null mice. CONCLUSIONS: Bombesin/GRP can induce features of BPD, including interstitial fibrosis and diminished alveolarization. GRPR appears to mediate all effects of GRP, but only part of the bombesin effect on alveolarization, suggesting that novel receptors may mediate some effects of bombesin in newborn lung.     

Calcium signaling and secretion in pituitary cells

An important trigger of hormone secretion from pituitary cells is a rise in cytosolic Ca²⁺ ([Ca²⁺]_i). Pituitary cells may modulate [Ca²⁺]_i by an increased membrane flux from the extracellular space and/or by a release from intracellular stores. Both mechanisms can support exocytosis, although in different pituitary cell types one or the other mechanism may predominate. Molecular events transducing a rise in [Ca²⁺]_i into hormone secretion are still poorly understood. Here, the exocytotic machinery in pituitary cells is briefly reviewed in terms of the spatial organization of [Ca²⁺]_i elevation relative to the Ca²⁺ sensor(s).     

Platelet-derived growth factor receptor independent proliferation of human glioblastoma cells: selective tyrosine kinase inhibitors lack antiproliferative activity

Purpose: The aim of this study was to investigate the role of platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) in the proliferation of human glioblastoma cells as a prerequisite for a new therapeutic approach to the treatment of malignant brain tumors with selective tyrosine kinase inhibitors such as imatinib. Methods and results: In the human glioblastoma cell lines U-87 MG, U-118 MG and U-373 MG different PDGF and PDGFR mRNAs were detected by RT-PCR, and the expression of the receptor proteins was demonstrated by immunostaining and flow cytometry. Moreover, functional activity of PDGFRs was demonstrated in PDGFRβ expressing glioblastoma cell variants by measuring the mobilization of intracellular Ca²⁺ upon PDGF-BB stimulation. However, addition of PDGF-BB to the serum-free culture medium had no stimulatory effect on cell proliferation. Furthermore, cell growth in serum-supplemented and serum-free medium was not affected by imatinib, leflunomide and AG-1296 at therapeutically relevant concentrations. Conclusion: Our results suggest that clinical antitumor effects of imatinib on glioblastoma, if any, are not mediated by the PDGFR.     

Lysophosphatidic acid and intracellular signalling in vascular smooth muscle cells

Growth of vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of atherosclerosis and hypertension. Lysophosphatidic acid (LPA), a natural phospholipid is thought to be an important VSMC mitogen and has recently been suggested to play an important role in the development of vascular disease. In the present study, we describe the effects of LPA on intracellular signalling pathways in VSMC. LPA (5 μg/ml) induced an increase of cytosolic free calcium concentration ([Ca²⁺]_i) in the presence and absence of extracellular Ca²⁺ and markedly stimulated the Na⁺/H⁺ exchanger. LPA dose-dependently caused a stimulation of the 42-kDa mitogen-activated protein kinase (MAP kinase) isoform with a maximum at 5 min. Also, LPA induced a 5-fold increase in [³H]thymidine incorporation into cell DNA above the basal value, as well as a 42% increase in cell number. Pretreatment of VSMC with pertussis toxin (PTX) (100 ng/ml) for 24 h markedly blunted the LPA-dependent intracellular signalling transduction including the increase in [Ca²⁺]_i, activation of the Na⁺/H⁺ exchanger, activation of MAP kinase and the increase in cell DNA synthesis. These findings demonstrate that the effects of LPA on intracellular signalling transduction pathway as well as on VSMC growth are mediated by PTX-sensitive guanosine triphosphate (GTP) binding protein (G_i protein).     

Characterization of the calcium response to thyrotropin-releasing hormone in lactotrophs and GH cells

Thyrotropin-releasing hormone (TRH) acts via a G-protein-coupled receptor on lactotrophs to increase the intracellular free calcium ion concentration, [Ca²⁺]_i. The [Ca²⁺]_i response depends on both TRH concentration and the duration of TRH exposure. An initial, short-lived [Ca²⁺]_i spike results from release of Ca²⁺ from intracellular stores, whereas a later sustained [Ca²⁺]_i increase, often characterized by [Ca²⁺]_i oscillations, results from an influx of extracellular Ca²⁺ through both voltage-gated and non-voltage-gated, store-operated Ca²⁺ channels. The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates at low doses. The mechanisms underlying the complex [Ca²⁺]_i response to TRH are discussed.     

Regulatory mechanism of bombesin on gastrin release

Bombesin has been demonstrated to stimulate gastrin release by an atropine-resistant mechanism. In the present study, the effects of truncal vagotomy and chemical sympathectomy on the gastrin release by exogenous and endogenous bombesin using rat antral mucosa in tissue culture were studied. Exogenous bombesin 10^-8 mol/l significantly stimulated gastrin release. The stimulation of gastrin release by bombesin was abolished by truncal vagotomy, but not altered by chemical sympathectomy. Bombesin antiserum inhibited gastrin release by blocking the effect of endogenous bombesin. The inhibition of gastrin release by bombesin antiserum was abolished by truncal vagotomy, but not altered by chemical sympathectomy. In addition, the concentrations of bombesin-like immunoreactivity in antral mucosa were not altered by truncal vagotomy. These results suggest that the mechanism of gastrin release by bombesin is influenced by non-cholinergic local nerves under vagal control.     

Agonist-specific Ca2+ signaling systems, composed of multiple intracellular Ca2+ stores, regulate gonadotropin secretion

Ca²⁺ signals regulate many cellular functions, including hormone secretion. Agonist-specific Ca²⁺ signaling may arise from the differential mobilization of multiple Ca²⁺ stores. Although they act through the same receptor subtype, two gonadotropin-releasing hormones (sGnRH and cGnRH-II) generate quantifiably different Ca²⁺ signals in goldfish gonadotropes, suggesting that their Ca²⁺-dependent signaling cascades may differ. We combined electrophysiology, Ca²⁺ imaging, and radioimmunoassay detection of gonadotropin (GTH-II) secretion to determine the role of intracellular Ca²⁺ stores in GnRH-stimulated exocytosis. Our findings suggest that voltage-gated Ca²⁺ channels do not mediate acute GnRH-signaling. Instead, both sGnRH- and cGnRH-II-stimulated GTH-II releases are dependent on Ca²⁺ mobilized from TMB–8/CPA-sensitive compartments. However, sGnRH, but not cGnRH-II, utilizes intracellular stores sensitive to caffeine and xestospongin C. We also identified a homeostatic mechanism where reduced extracellular Ca²⁺ availability increase GTH-II release by mobilizing Ca²⁺ stores. Our results are the first to suggest that several classes of intracellular Ca²⁺ stores differentially participate in agonist signaling and homeostasis in gonadotropes.     

Comparative effects of bombesin and porcine gastrin-releasing peptide in the dog

Bombesin and porcine gastrin-releasing peptide (GRP) share a common C-terminal decapeptide fragment and are known to have similar bioactivity. The potencies of parenterally administered GRP and bombesin have been compared in their effects on gastric acid output, serum gastrin, and serum pancreatic polypeptide. In the dose range 0–720 pmol/kg/hr, infusions of both peptides resulted in dose-related increases of gastric acid output and corresponding elevations of serum gastrin and pancreatic polypeptide. At the dose of 1440 pmol/kg/hr, both bombesin and GRP resulted in a decrease in gastric acid output and serum gastrin. The mechanism of this inhibition is unknown. No significant difference in potency between the two peptides in changing gastric acid output, serum gastrin, and pancreatic polypeptide was observed. The demonstration of equimolar potency of porcine GRP and bombesin support the concept that GRP is mammalian bombesin.This study was supported by the National Health and Medical Research Council of Australia.     

Stimulation of growth hormone and prolactin release from rat pituitary cell aggregates by bombesin- and ranatensin-like peptides is potentiated by estradiol, 5 alpha-dihydrotestosterone, and dexamethasone

The effect of the bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-C (NMC), and the ranatensin-like peptides, neuromedin-B (NMB), neuromedin-B30 (NMB30), and neuromedin-B32 (NMB32), on pituitary GH and PRL release was studied in perifused anterior pituitary aggregate cell cultures from 9- to 12-week-old male rats cultured in serum-free defined medium supplemented with 0.05 nM T3 and 4 nM dexamethasone (DEX). All peptides stimulated PRL and GH release. GRP and NMC stimulated hormone release in a concentration-dependent manner between 0.1-10 nM. NMB was slightly more potent than NMB30 and NMB32, but was significantly less potent than GRP and NMC. The magnitude of the PRL response to GRP and NMC inversely correlated with that of the GH response. Cultures with relatively low PRL response levels displayed high GH responses, whereas the opposite was found in cultures with high PRL response levels. The stimulatory actions of GRP, NMC, and NMB were blocked by the bombesin receptor antagonist Leu13 psi (CH2NH) Leu14-bombesin, supporting the specificity of the findings. Addition of 1 nM estradiol (E2) to the culture medium provoked an impressive (4- to 10-fold) increase in the magnitude of the GH response to NMC without changing the EC50 value (0.5 nM). In contrast, E2 significantly decreased the stimulation of GH release by rat GH-releasing factor. In the E2-treated aggregates 3 nM NMC stimulated GH release to a comparable extent as 0.1 nM GRF. 5 alpha-Dihydrotesterone (10 and 100 nM) and DEX (80 nM) also enhanced the GH response to NMC, but to a much smaller extent than E2. E2 had also a stimulatory effect on the PRL response to NMC, particularly in cultures with a low intrinsic PRL response. The PRL response to NMC was decreased by DEX and slightly augmented by 5 alpha-dihydrotestosterone. It is concluded that bombesin- and ranatensin-like peptides have a stimulatory effect on GH and PRL release at the pituitary level. Since their action on GH release is strongly potentiated by E2 and much less so by glucocorticoids, these peptides clearly distinguish their activity and specificity from that of the protagonist releasing factor GH-releasing factor, suggesting a role in sex-related differences in GH release or in the control of GH secretion during sexual maturation.     

The modulation of placental lactogen release by opioids: a role for extracellular calcium

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 μg/ml streptomycin. ⁴⁵Ca²⁺ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of ⁴⁵Ca²⁺ influx by ethylketocyclazocine (EKC) with an EC₅₀ of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50, 488 (kappa agonist) had the same stimulatory effect as EKC (221 ± 25% of control). D-Ala²,NMe-Phe⁴,Gly-ol⁵)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca²⁺ influx (128 ± 5% of control, p > 0.05) whereas D-Ser²,Leu, Thr⁶)-enkephaIin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced ⁴⁵Ca²⁺ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins. The role of extracellular calcium in the regulation of hPL release by kappa opiates was confirmed by experiments in which ionomycin (ionophore) and Bay K 8644 (calcium-channel agonist) mimicked the stimulatory effect of EKC on hPL release. Also, without extracellular Ca²⁺, the basal (control) and EKC-stimulated hPL release were significantly lower than with 1.8 mM Ca²⁺. Moreover, nifedipine (calcium-channel antagonist) inhibited both basal and EKC-stimulated hPL release. These results suggest that calcium influx is one mechanism mediating opioid-stimulated hPL release by human term trophoblastic cells.     

Estrogen receptor independent rapid non-genomic effects of environmental estrogens on [Ca2+]i in human breast cancer cells

The aim of this study was to identify and characterize an alternative pathway through which environmental estrogenic compounds may mediate their intracellular effects. Three human breast cancer cell lines were employed including MCF-7 cells, which express both ER and ERβ; MDA-MB-231 cells, which express ERβ but not ER; and SKBR-3 cells, which express neither ER nor ERβ. The effect of environmental estrogenic compounds on intracellular calcium ion concentration ([Ca²⁺]_i) was measured and compared to that of 17β-estradiol (E2). A rapid and maintained increase in [Ca²⁺]_i was observed following the application of nanomolar concentrations of environmental estrogens and E2 regardless of the expression of ER and ERβ. Removal of extracellular Ca²⁺ completely abolished the steroid-induced [Ca²⁺]_i increase. Pre-treatment of cells with the estrogen receptor (ER) antagonist ICI 182,780 had no effect on either basal [Ca²⁺]_i or the steroid-triggered [Ca²⁺]_i response.

In summary, we have demonstrated ER independent rapid non-genomic effects of environmental estrogenic compounds, at nanomolar concentrations, on [Ca²⁺]_i. The results of this study demonstrate an alternative pathway to explain potent intracellular effects of endocrine disrupting chemicals.     

Tranilast inhibits contraction and Ca movement of porcine coronary arteries

In a recent clinical study, tranilast, an anti-allergic agent, was shown to reduce the rate of coronary restenosis after percutaneous transluminal coronary angioplasty, although the mechanism of this effect is unclear. The present study was undertaken to investigate the effects of tranilast on contraction and Ca²⁺ movement of the coronary arteries. We characterized the effects of tranilast on isometric force and aequorin-estimated intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of porcine coronary artery strips. Tranilast concentration-dependently (10–500 μM) inhibited histamine (3×10⁻⁵ M)-induced contraction of the coronary arteries. A similar tendency was observed in the response to high K⁺ (30 mM) stimulation. Histamine caused phasic and tonic increases in [Ca²⁺]_i, and high K⁺ caused a tonic increase in [Ca²⁺]_i of smooth muscle, both of which were significantly suppressed in the presence of tranilast. These results suggest that tranilast inhibits the contraction of coronary arteries by inhibiting both Ca²⁺ influx from extracellular environment and Ca²⁺ release from intracellular Ca²⁺ stores, which might be related to its preventive effect on restenosis after coronary angioplasty.     

Increased function of the voltage-dependent calcium channels, without increase of Ca release from the sarcoplasmic reticulum in the arterioles of spontaneous hypertensive rats

It has been reported that the increased function of the voltage-dependent calcium channels (VDCC) in the artery is involved in the increase of peripheral resistance in hypertension, and that the sarcoplasmic reticulum (SR) in the artery plays an important role in preventing the development of hypertension via a buffering effect. However, no reports have described the role of VDCC and SR in resistance arterioles in the development or maintenance of hypertension. We investigated the function of VDCC and of SR in the cremaster arterioles of spontaneous hypertensive rats (SHR) and age-matched Wistar Kyoto rats (WKY). The changes in diameter and the intracellular calcium ion concentration ([Ca²⁺]_i) in the microdissected arterioles, using fluorescent dyes, were measured with videomicroscopy. The KCl concentration-response curves were analyzed in 4- to 5- and 7- to 8-week-old SHR and WKY. The changes in the vascular diameter and [Ca²⁺]_i in response to ryanodine, an -1 adrenoceptor, and angiotensin-II stimulation were compared between the 7- to 8-week-old SHR and WKY. We found an increase in the Ca²⁺ influx by VDCC in the early hypertensive stage, but not in prehypertensive SHR. However, after the onset of hypertension, there were no significant differences from WKY in the SR function mediated by Ca²⁺-induced Ca²⁺ release or inositol 1,4,5-trisphosphate-induced Ca²⁺ release. In conclusion, an increased influx of Ca²⁺ in the cell membrane, without a buffering effect of SR, was associated with progression of hypertension in the cremaster arterioles of SHR.     

Potentiators of insulin secretion modulate Ca sensitivity in rat pancreatic islets

Insulin secretion stimulated by 10 mM glucose was potentiated by forskolin, an activator of adenyl cyclase, by acetyl choline which may enhance turnover of inositol phospholipids, and by tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. None of these agents initiated insulin secretion in the presence of 2 mM glucose. Glucose-stimulated insulin secretion was markedly dependent on the concentration of extracellular Ca²⁺: at or below 10 μM Ca²⁺ no insulin secretion was evoked by glucose in freshly isolated islets. The threshold Ca²⁺ requirement was increased after culture of islets for 44 h. In both fresh and cultured islets the presence of a potentiator of secretion produced both a marked increase in the maximum rate of glucose-stimulated insulin secretion and a lowering of the requirement for extracellular Ca²⁺. Thus potentiation of insulin release involves an increase in the sensitivity of the B cell to Ca²⁺.     

Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal

AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate(InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor(InsP3R3) in ICC.Protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85%(P 0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05%(P 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin(5 μmol/L) prevented CCK-8S(100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C(1 μmol/L) could also abolish the CCK-8S-induced effect,indicating that Ca2+ release from InsP3R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC.On the other hand,by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine,a smaller but significant rise in the [Ca2+]i could be still elicited by CCK-8S.These data suggest that the [Ca2+]i release is not stimulated or activated by the influx of extracellular Ca2+ in ICC,but the influx of extracellular Ca2+ can facilitate the [Ca2+]i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3,which could be prevented by chelerythrine.Pretreatment with lorglumide(5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group,treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA(10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of100 nmol/L PMA was most obvious.Likewise,the effect of CCK-8S was augmented by the pretreatment with chelerythrine(10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect.CONCLUSION:CCK-8S increases [Ca2+]i in ICC via the CCK1 receptor.This effect depends on the release of InsP3R-operated Ca2+ stores,which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.     

Diazepam enhances hypericin-induced photocytotoxicity and apoptosis in human glioblastoma cells

Glioblastoma multiforme (GBM) is neoplasm which is resistant to all currently used treatment modalities including surgery, radiation therapy and chemotherapy. Photodynamic therapy (PDT) has been suggested as a novel therapeutical approach to the treatment of malignant gliomas. Here, we attempted to enhance hypericin-induced photocytotoxicity and apoptosis by diazepam, a non-selective ligand of peripheral benzodiazepine receptors (PBR) which seem to play an important role in apoptosis regulation. For the study, we used U-87 MG and U373 MG glioma cell lines and primary cultures of GBM cells prepared from peroperatively obtained tumor specimens. The patients included 7 histologically confirmed GBMs. Colorimetric MTT assay was employed to study the photocytotoxic effects of hypericin and diazepam. Flow cytometry was used to detect apoptosis and assess the proapoptotic effects of diazepam. We found that hypericin upon photoactivation exerts strong cytotoxic effects against U-87 MG and U373 MG cells as well as primary GBM cell cultures. No cytotoxic effect of hypericin was observed under dark conditions. Diazepam inhibited cell growth in U-87 MG cells and primary cultures whereas proliferation of U373 MG cells remained unaffected. When hypericin was combined with diazepam, photocytotoxicity was increased in U-87 MG cells and primary cultures unlike U373 MG cells. Flow cytometric analysis revealed photoactivated hypericin-induced apoptosis in both cell lines. Apoptosis was significantly enhanced by diazepam in U-87 MG cells. However, no such effect was observed in U373 MG cells. In the present study, we showed that photocytotoxic effect of hypericin in glioma cells can be potentiated by diazepam. This effect is underlied by the ability of diazepam to facilitate hypericin-induced apoptosis. This work provides support to performe clinical studies involving diazepam in the antiglioma treatment regimens as an apoptosis-modulating agent.     

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach.     

Bombesin-like peptides and mast cell responses: relevance to bronchopulmonary dysplasia?

Bombesin-like peptides (BLPs) are elevated in newborns who later develop bronchopulmonary dysplasia (BPD). In baboon models, anti-BLP blocking antibodies abrogate BPD. We now demonstrate hyperplasia of both neuroendocrine cells and mast cells in lungs of baboons with BPD, compared with non-BPD controls or BLP antibody-treated BPD baboons. To determine whether BLPs are proinflammatory, bombesin was administered intratracheally to mice. Forty-eight hours later, we observed increased numbers of lung mast cells. We analyzed murine mast cells for BLP receptor gene expression, and identified mRNAs encoding bombesin receptor subtype 3 and neuromedin-B receptor (NMB-R), but not gastrin-releasing peptide receptor. Only NMB-R-null mice accumulated fewer lung mast cells after bombesin treatment. Bombesin, gastrin-releasing peptide, NMB, and a bombesin receptor subtype 3-specific ligand induced mast cell proliferation and chemotaxis in vitro. These observations support a role for multiple BLPs in promoting mast cell responses, suggesting a mechanistic link between BLPs and chronic inflammatory lung diseases.     

Calcium and lipoprotein lipase synergistically enhance the binding and uptake of native and oxidized LDL in mouse peritoneal macrophages

The influence of Ca²⁺ and Mg²⁺, together with lipoprotein lipase (LPL), on the binding and uptake of Eu³⁺-labeled native and oxidized low density lipoprotein (LDL) to mouse peritoneal macrophages (MPM), and on the deposition of esterified cholesterol in these macrophages, were studied. We found that both LPL and Ca²⁺ (but not Mg²⁺) increased the binding and uptake of native and mildly or moderately oxidized LDL, and the subsequent deposition of cholesterol esters in MPM. When added together, LPL and Ca²⁺ synergistically increased the binding and uptake of native and oxidized LDL, and the deposition of esterified cholesterol derived from native and mildly or moderately oxidized LDL, in MPM. Since both calcium and LPL are found in the atherosclerotic lesions, our results suggest that Ca²⁺ and LPL may synergistically promote foam cell formation and atherogenesis. Furthermore, future research in the metabolism of lipoproteins should take into account the calcium levels in the experimental conditions.